Selective protein phosphorylation is a mechanism of major importance for homeostatic regulation in mammalian cells and is central to the action of a wide variety of effectors including cyclic AMP, cyclic GMP, insulin, and Ca2 ion. Nevertheless, information about the intracellular substrates for effector-mediated phosphorylation and regulation of their phosphorylation is scanty. We have recently developed a new approach for studying protein phosphorylation in intact cells using high resolution two-dimensional gel electrophoresis to separate phosphorylated from nonphosphorylated forms of substrate proteins. A study of the responses of S49 mouse lymphoma cells to elevation of intracellular cyclic AMP (Steinberg, R.A. and Coffino, P. (1979) Cell 18, 719-733) has illustrated the utility of this approach and revealed unexpected complexity in cyclic AMP-mediated responses. We propose to extend this work using detailed kinetic analyses, phosphate turnover studies, phosphopeptide analysis, and in vitro phosphorylation to obtain a more complete understanding of the interaction between components of the protein phosphorylation system in intact cells. We also plan studies comparing cyclic AMP-mediated phosphorylations in a variety of differentiated cell types, comparing substrates for phosphorylations mediated by a variety of effectors, and assessing interactions between effectors at the level of phosphorylation substrates.